This invention relates to surface plasmon resonance (SPR) sensors for use in biological, biochemical and chemical testing. The invention is particularly concerned with adaptions of such sensors to enable larger areas to be covered and thus enable the SPR technique to be used, for example, in applications such as sequencing.
Sequencing of macromolecules, for example nucleic acids such as DNA, is usually carried out by biochemical fragmentation, followed by gel electrophoresis. Following electrophoresis, the sequence is detected by exposure of the gel to autoradiographic film to give a two-dimensional picture from which the sequence of individual nucleotide bases can be read. There are currently two main techniques used for fragmentation, the Maxam-Gilbert method involving the cleavage of DNA molecule by a two-stage addition of chemicals, and the Sanger method involving the preparation of separate samples of dideoxynucleotide of each of the four bases C, G, A and T and then the growth of a complementary DNA strand to that of the DNA under test using an enzyme. Both techniques end up with four different sub-samples, enabling identification of the four bases, each containig fragments of different lengths. A radioactive or fluorescent label is attached to each fragment to enable detection of the sequence using autoradiography. In electrophoresis, the four different sub-samples, containing respective sets of labelled fragments are each placed in a separate well and thus generate a separate and distinct track across the gel as the electrophoresis proceeds. After the electrophoresis has finished, the gel is separated and typically dried and is then, in the case of the radioactive label, exposed to autoradiographic film to obtain an image of the pattern of labelled fragments of different length and belonging to different nucleotide groups.
Autoradiography, while it can offer good sensitivity and resolution is an inconvenient technique, taking considerable skill to achieve good results, and is a fairly lengthly procedure, requiring many hours or even several weeks of exposure. Furthermore, separation of the gel after electrophoresis can lead to damage unless great care is taken, and the very necessary drying of the gel prior to exposure is also tricky since shrinking of the gel must be avoided if meaningful results are to be obtained.
In the present invention, the requirement for autoradiography is eliminated by enabling direct monitoring of the gel using a surface plasmon resonance (SPR) detector. The SPR detector operates to detect changes in the refractive index of the gel across the gel surface. Areas of the gel where fragments are present will have a different refractive index to those where they are not and a "picture" of the gel surface can thus be built up. Although the use of gel will be assumed in this specification, the technique can in fact be used with gel-less and other forms of capillary electrophoresis.
Monitoring can be in "real time"--i.e. as the electrophoresis test proceeds, or can be carried out on the gel after electrophoresis has been completed. In this latter case it may be advantageous to expose the gel to air after electrophoresis has finished so that the gel shrinks and thus concentrates the fragments into a smaller area which can be examined by the SPR detector.
The use of surface plasmon resonance in connection with biosensors has been described in detail in our copending European patent application Nos. 0305109 and 89300544.7, together with our copending British patent application Nos. 8811053.1, 8811054.9, 8811919.3 and 8813307.9.
All of the techniques described in these applications can be applied to the teaching of the present invention.
Surface plasmon resonance is the oscillation of the plasma of free electrons which exists at a metal boundary. These oscillations are affected by the refractive index of the material adjacent the metal surface and it is this that forms the basis of the sensor mechanism used in the present invention. Surface plasmon resonance may be achieved by using the evanescent wave which is generated when a p-polarized light beam is totally internally reflected at the boundary of a medium, e.g. glass, which has a high dielectric constant. A paper describing the technique has been published under the title "Surface plasmon resonance for gas detection and biosensing" by Lieberg, Nylander and Lundstrom in Sensors and Actuators, Vol. 4, page 299. Illustrated in FIG. 1 of the accompanying drawings is a diagram of the equipment described in this paper. A beam 1 of light is applied from a laser source (not shown) onto an internal surface 2 of a glass body 3. A detector (not shown) monitors the internally reflected beam 4. Applied to the external surface 2 of glass body 3 is a thin film 5 of metal, for example gold or silver, and applied to the film 5 is a further thin film 6 of organic material containing antibodies. A sample 7 containing antigen is brought into contact with the antibody film 6 to thus cause a reaction between the antigen and the antibody. If binding occurs, the refractive index of the layer 6 will change owing to the size of the antibody molecules and this change can be detected and measured using the surface plasmon resonance technique, as will now be explained.
Surface plasmon resonance can be experimentally observed, in the arrangement of FIG. 1, by varying the angle of the incident beam 1 and monitoring the intensity of the internally reflected beam 4. At a certain angle of incidence the parallel component of the light momentum will match with the dispersion for surface plasmons at the opposite surface 8 of the metal film. Provided that the thickness of metal film 5 is chosen correctly there will be an electromagnetic coupling between the glass/metal interface at surface 2 and the metal/antibody interface at surface 8 as a result of surface plasmon resonance, and thus an attenuation in the reflected beam 4 at that particular angle of incidence. Thus, as the angle of incidence of beam 1 is varied, surface plasmon resonance is observed as a sharp dip in the intensity of the internally reflected beam 4 at a particular angle of incidence. The angle of incidence at which resonance occurs is affected by the refractive index of the material against the metal film 5--i.e. the antibody layer 6--and the angle of incidence corresponding to resonance is thus a direct measure of the state of the reaction between the antibody and the antigen. Increased sensitivity can be obtained by choosing an angle of incidence half way down the reflectance dip curve, where the response is substantially linear, at the beginning of the antibody/antigen reaction, and then maintaining that angle of incidence fixed and observing changes in the intensity of the reflected beam 4 with time.
A typical basic detector comprises the following components:
1) A source of electromagnetic radiation, most likely in or near the visible light region; PA1 2) A block of transparent material such as glass on one surface of which is applied a thin film of metal, for example silver or gold, and on which is supported a medium to be tested in such a way that the metal film is sandwiched between the medium and the glass block; PA1 3) Means for directing the electromagnetic radiation from the source into the transparent block in such a way that the radiation is subject to total internal reflection at that surface of the block to which the metal film is applied; PA1 4) A detector for monitoring the intensity of the radiation which is totally internally reflected. PA1 1. Immersion in molten metal nitrates and other molten salts. This has the effect of introducing ions into the surface in a manner which can be structured and which can act as foci for island formation. PA1 2. Ion bombardment of cold or hot glass to introduce nucleating sites. The removal of the more mobile ions has been demonstrated to reduce the thickness at which the evaporated film becomes continuous. PA1 3. Electroless plating or electroplating over lightly evaporated films (0 to 100 angstroms thick). Electroless plated films survive to a greater thickness than evaporated films and could form more stable nuclei for subsequent coating. PA1 4. Evaporating onto electroless plated films. The electroless plated films have a stronger tendency to an island structure and to bigger islands with greater spacing than evaporating films. This could be of advantage in tuning to light of a prescribed wavelength. PA1 1. Controlling the surface temperature of the transparent plate during coating. Using a higher temperature substrate increases the islands' size and the spacing between them and conversely. PA1 2. Evaporating in the presence of a magnetic or electrostatic field or electron emission device to control the ion content of the vapour stream. The state of charge of the substrate is know to affect the island structure. PA1 3. Controlling the angle of incidence of the evaporated vapor stream relative to the surface of the plate. The mobility of the evaporated atoms and hence their ability to form bigger islands is greater when the momentum of the atoms relative to the glass surface is increased.
The point of incidence of the incoming beam of radiation defines a small sensitive zone centered around the point. As the refractive index of the medium changes, the angle of incidence at which SPR occurs changes and, for a fixed angle of incidence, this manifests itself as an alteration in the intensity of the totally internally reflected beam, as detected by the detector. A significant improvement in signal/noise ratio can be achieved, however, by monitoring the entirety of the dip resulting from surface plasmon resonance. This may be achieved either by rapidly scanning the incoming beam of radiation across the angles of incidence which result in surface plasmon resonance, such as described in our application No. 8811053.1, or by utilizing the so called "fan beam" as described in our copending European patent application No. 0305109. In a variant of the fan beam, a line point of incidence is formed using a wedge beam, possibly in conjunction with a semicylinder, as described in the aforementioned European patent application.